Hydrocarbons, including petroleum based hydrocarbons, can be released into the environment from industrial processes through discharges and emissions, from surface spills such as open water spills, above ground storage tank overfilling and releases, from oil spills and tanker purging during ship transport, from oil drilling and exploration operations, from agricultural operations such as cropland weed and pest management associated with chemical applications, and from subsurface releases such as leaking underground storage tanks, undesirably polluting the environment. "Petroleum based hydrocarbons" is understood to include by way of example, and not as a limitation to the present invention, chemicals toxic to the environment such as crude oil, distilled or refined fuels, halogenated pesticides, halogenated herbicides, halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and/or methane and the like. Release of such toxic chemicals is referred to herein as a "contaminant". The environment into which the contaminant is released, be it liquid or solid or gaseous media, includes by way of example, and not as a limitation to the present invention, surface water, ground water, wells, rivers, estuaries, the ocean, surface sediment, subsurface soil, surface soil, foliage and/or soil containing natural plant growth, and/or air, and the like and is referred to herein as "contaminated media".
Oil spilled at sea often reaches the littoral zone and becomes coated on or mixed with soil, or other solids, such as rocks, and vegetation. Oil adsorption and absorption on or in these materials renders the petroleum contaminants less mobile and difficult to clean up or remediate using conventional physical/mechanical remediation techniques, such as filtering, excavation or mechanical removal. Such techniques also tend to adversely affect the ecosystems in proximity to the contaminants.
Contaminants discharged from industrial processes are often mixed as an aqueous influent to treatment systems and then as an effluent to receiving waters. These chemicals are also adsorbed on suspended solids in water and can settle out and thus become incorporated into the sediment. Once settled in the sediment, the contaminants become less mobile and difficult to remediate using conventional physical/mechanical remediation techniques without causing damage to surrounding ecosystems.
Herbicides and pesticides used in cropland management are applied to plants and released in spills or as excess in over application. These applications and releases are washed into the soil and plants from irrigation practices or from atmospheric precipitation. Such herbicides and pesticides accumulate in the rhizosphere where conventional physical/mechanical remediation techniques also prove difficult or detrimental to the surrounding ecosystem.
Distilled or refined fuels, including chemical mixtures such as gasoline and diesel fuels stored in above ground or underground storage tanks released to the environment from overspills during tank filling and tank and piping failures are adsorbed or absorbed respectively into the soil and plants adjacent to such releases. There they tend to become less mobile and/or accumulate on or in the groundwater resulting in dissolved aqueous and non-aqueous phase liquids containing these chemical contaminants. Further migration of these chemicals also results in widespread aquifer contamination. Such contaminated media are also difficult to remediate and can harm human health and sensitive ecosystems.
Bioremediation of these contaminants tends to be the best technology and most cost effective means to remediate such contaminated media. What is meant by "bioremediation" or "bioremediation activity" is the microbial oxidation and/or mineralization (i.e., biodegradation) of contaminants. "Mineralization" means the bioconversion of contaminants to carbon dioxide, water and occasionally new microbial cell growth. "Detoxication" (sometimes designated "detoxification") refers to the change in a contaminant (molecule or complex mixture) that renders it less harmful to one or more susceptible organisms (i.e. microorganisms, plants, animals or humans). See M. Alexander, Biodegradation and Bioremediation, pp. 41-48 (1994), incorporated herein by reference.
Many naturally occurring microorganisms are useful in microbial oxidation and mineralization of such contaminants because they generate enzymes that oxidize and mineralize the contaminants through what is known metabolically as catabolism. However a disadvantage of conventional microbial oxidation and mineralization is that the rate of biochemical oxidation (i.e., bioremediation) of the contaminated media is often undesirably slow. One reason for the slow bioremediation rate of the contaminants is that the microbes may exist only in small amounts naturally in the contaminated media. It has also been purported that useful indigenous microorganisms tend to be attacked by predatory microorganisms, thereby keeping the amount of indigenous microorganisms relatively low. In addition, naturally occurring microorganisms may only produce small amounts of the enzymes required for oxidation and mineralization thereby also causing slow oxidation and mineralization rates of the contaminants. It is believed that the rate of enzyme production occurs at low rates and can be ultimately inhibited through a cellular phenomenon known as catabolite repression.
It is known that microorganisms tend to exhibit decreased production of enzymes useful in oxidizing and mineralizing contaminants when insulted with toxic contaminants thereby decreasing or stopping the rate of biodegradation of such contaminants. These reasons, either combined or independently, render microbial biodegradation of contaminated media undesirably slow and/or incomplete to mineralization.
Genetically engineered microorganisms (GEMs) have been developed in an attempt to overcome some of the above problems with naturally occurring microorganisms. GEMs however tend to be unacceptable to environmental regulatory authorities for widespread and uncontrolled environmental application due to their uncertain effects on the environment.
There are numerous enzymes present in microorganisms known to catalyze the biodegradation of the contaminants. In bacteria, cAMP has been found to play a role in the formation of the constitutive enzymes necessary to catalyze the breakdown of secondary sugars such as galactose and arabinose in the presence of glucose. However, enzymes known to be associated with the break down of sugars, such as glucose, lactose and galactose, and not those associated with the oxidation and mineralization of toxic contaminants tend to exhibit cAMP induced activity. For example, H. V. Rickenberg, Cyclic AMP in Prokaryotes, pp. 353-369 (1974), ("Rickenberg"), describes the synthesis of several proteins (enzymes) controlled by cAMP. It has been demonstrated that cAMP is required for the effective synthesis of beta-galactosidase and tryptophanase in E. Coli. Rickenberg further describes that in "E. Coli exogenous cAMP overcomes both the severe transient (citation omitted) and less severe steady-state catabolite repression (citation omitted) of the synthesis of beta-galactosidase caused by the presence of glucose in the medium." H. V. Rickenberg, Cyclic AMP in Prokaryotes, pp. 354-355.
It was found that inhibition of biolumenescence of Photobacterium phosphoreum is a useful measure of general toxicity associated with solids including soil. K. K., Kwan, Direct Solid Phase Toxicity Testing Procedure, 8 Environ. Toxicol. Water Qual., 345, (1993), incorporated herein by reference. The MICROTOX.RTM. solid phase test kit is based on the inhibition of the biolumenescence enzyme system associated with the test marine bacterium (Photobacterium phospherum). See also M. W. Greene, et al., Measurement of Soil and Sediment Toxicity to Bioluminescent Bacteria When In Direct Contact for a Fixed Time Period, 53-63, Proc. 65.sup.th Annu. Conf. & Expos., New Orleans, La., Sep. 20-24, 1992; K. K. Tung, et. al., The Solid Phase Assay: New Microtox Test Procedure, Proc. 17.sup.th Annu. Aquatic Toxicity Workshop, November 5-7, Vancouver B.C., Vol. 1, 1991.
Moreover, it is known from soil ecotoxicology that the de novo biosynthesis of beta-galactosidase induction is inhibited by the presence of a toxic xenobiotic and has been correlated to a decrease in biomass production in plants. Toxicity tests in soil have been based on such enzyme inhibition phenomena. For example, the direct solid phase toxicity testing procedure which uses the Toxi-Chromotest kit is based on inhibition by chemicals of the de novo biosynthesis of beta-galactosidase. K. K., Kwan, Direct Toxicity Assessment of Solid Phase Samples Using the Toxi-Chromotest Kit, 8 Environ. Toxicol. Water Qual., 223, (1993). Therefore it is known that the presence of toxic xenobiotics in soil tends to inhibit certain enzyme activity responsible for biodegradation.
According to M. Alexander, Biodegradation and Bioremediation, pp. 36-40, (1994), ("Alexander"), incorporated herein by reference, constitutive enzymes are produced by certain microorganisms regardless of whether substrates for those enzymes are present. By contrast, inducible enzymes are only formed in appreciable amounts when the substrate, or a structurally related compound, is present. Induction is known to be a complex process and involves particular substrates which induce or increase the rate of formation of certain degradative enzymes. However, because xenobiotics, more particularly, petroleum based hydrocarbons, are known to inhibit certain enzyme activity responsible for biodegradation, constitutive enzymes known to catalyze the breakdown of sugars such as glucose, lactose and arabinose are often ineffective for bioremediating contaminants which are structurally different than sugars, and which also require inducible enzymes for their degradation.
In addition, the products of catabolism (catabolites) can act as repressors inhibiting the formation of inducible enzymes. This inhibition is known as "catabolite repression" and according to Alexander, controls the enzymatic population where products generated during catabolism of one substrate can repress the synthesis of enzymes that function to degrade a second substrate. Catabolite repression has been implicated in diauxie growth and usually occurs where a preferred energy source, such as carbon from sugar is present and catalyzed first which accordingly represses catabolic inducible enzymes required for the biodegradation of complex contaminants.
Recent research indicates that cAMP may also play a role concerning signal transduction with the plant hormone auxin. See Ichikawa, et al., Identification and Role of the Known Adenyl Cyclase In Auxin Signaling and Higher Plants, Nature, Vol. 390 (6661), December 18/25 (1997) pp. 689-701 ("Ichikawa"). Ichikawa discloses findings relating to tobacco protoplasts expressing the enzyme adenylyl cyclase which produces cAMP. Ichikawa discloses that the cAMP generated by the tagged tobacco protoplasts can replace auxin in triggering its cell division, and suggests that cAMP may be part of the auxin signal-transduction pathway. However, it is also known that there are toxic effects associated with high levels of auxin and that many herbicides are synthetic auxins which can inhibit the growth of plants. See U.S. Pat. No.4,919,702 to Weltzien et al., hereby incorporated by reference. A chemical is considered "phytotoxic" when its presence reduces the growth or alters normal development of plants. Thousands of organic chemicals termed "phytotoxins" have such properties.
Forskolin, also called colforskoli, a diterpene, induces cAMP formation. Forskolin binds to the catalytic site of adenylate cyclase which is the enzyme responsible for the formation of cAMP in the microbial cell. At the concentration of about 10 micromole/liter, forskolin causes an increase in the cellular concentration of cAMP by activating adenylate cyclase. Yet forskolin is also known to be useful in the treatment of glaucoma. U.S. Pat. No. 4,476,140 to Yale University describes the use of forskolin in the treatment of glaucoma in mammals, hereby incorporated by reference. Forskolin, as does cAMP, possesses vasodilating and cardiostimulating properties, apparently due to its basic ability to stimulate the adenylate cyclase and thus increase the cellular concentration of cAMP in mammals. See U.S. Pat. Nos. 4,088,659, and 4,476,140, hereby incorporated by reference.
A disadvantage to the prior art of remediating pollutants contained in contaminated media is that often the nature of the media, and the extent of its contamination requires the application of bioremediation techniques in order to prevent concomitant damage to the surrounding ecosystems. The dilemma has been that the very nature of such toxic contaminants, like petroleum based hydrocarbons, inhibit enzymatic induced catabolism known to be responsible for metabolic degradation of xenobiotics. Moreover, enzymes known to be effective for the degradation of simple sugars, or believed to be linked to the regulation of certain plant cell division have not been shown to be useful or effective in oxidizing and mineralizing xenobiotic contaminants, detoxifying contaminated media or promoting the growth of such plants in the presence of such contaminants.
What is desired, therefore, is a composition comprising a mixture of microorganisms and compounds which result in the rapid oxidation, detoxification, and mineralization of contaminants that can be applied to contaminated media that have been previously difficult to remediate using conventional techniques. What is further desired is a mixture of microorganisms and compounds, wherein the mixture results in a more rapid oxidation and mineralization of contaminants by the microorganisms than without the compounds, thereby facilitating bioremediation and detoxication of contaminated media and also resulting in enhanced plant growth in such contaminated media, and methods for making and using such compositions.